Abstract A universal influenza vaccine is believed to be possible if conserved regions of influenza are effectively targeted and appropriate immune responses are generated against those targets. The enhanced safety, stability, and accelerated product development and production generally provided by DNA vaccination make it an appealing approach to develop such a universal influenza vaccine. Unfortunately, immune responses to universal influenza antigens are typically weak and earlier studies of DNA vaccination in humans showed poor humoral immunogenicity. However, recent advancements in vaccine delivery and use of adjuvants and viral vector boosts have improved the outlook of DNA vaccination. To overcome obstacles to developing an effective, practical, and truly universal influenza vaccine, we propose to further develop and test our DNA prime / viral-vectored boost vaccine regimen, which employs novel immunogens derived from the following four conserved influenza A antigens: 1) the stem region of hemagglutinin (Stem); 2) the matrix 2 protein ectodomain (M2e); 3) the nucleoprotein (NP); and 4) the matrix 1 protein (M1). Our phase I results indicate that together, these antigens evoke the immunological breadth and types of immune responses necessary to target a broad range of both seasonal and potential pandemic influenza strains and to evoke appropriate antiviral immune responses necessary to combat these influenza viral infections. Our strategy also uses a clinically proven DNA adjuvant that maximizes immunogenicity, tunes the responses toward an antiviral Th1 phenotype, and improves the anti- viral activities of the humoral immune response. Under this phase II SBIR application, we will test a viral-vectored boost for amplification of the DNA-raised immune responses. We will also determine the protective effects of our vaccine in other relevant preclinical animal models (ferrets and macaques) and perform studies intended to further define the vaccine's mechanisms of action. 3